1. Field of the Invention
The present invention relates to novel compounds, and more particularly to novel assay reagents suitable for use, inter alia, in the detection and measurement of catalytic activity from an enzyme or polypeptide pair, natural or synthetic, having the catalytic activity of an enzyme in the analysis of various compounds in biological fluids or the like.
2. Description of the Prior Art
For a variety of clinical purposes such as, for example, monitoring dosage schedules, monitoring hormone levels, checking for recent ingestion or following pharmacological dynamics of bioavailability, absorption, degradation or excretion, it is a great advantage to measure the concentration of various drugs or the like to the nanomolar or even picomolar level. As is known, radioimmunoassay can accomplish analyses of this type. To carry out an analysis, an acceptable kit or system must include an antiserum, a standard or known concentrations of the compound (i.e.,--analyte) to be measured, a radiolabeled derivative of the compound to be measured, and a buffering agent or agents. The antiserum is produced by bleeding animals which have been immunized by innoculation, for example, with the hapten-protein conjugate (immunogen) corresponding to the compound to be measured.
As is well known, the technique of radioimmunoassay, in general, measures the competition between radioactively labeled analyte and unlabeled analyte for binding sites on the antibody in the antiserum. By adding to the antiserum known amounts of the analytes to be assayed and a radiolabeled analog, a dose-response curve for bound or free analyte versus concentration of analyte is constructed. After this immuno-calibration has been carried out, unknown concentrations can then be compared to the standard dose-response curve for assay. Crucial to this type of assay is the existence of radioactive analytes which compete effectively with non-radioactive analytes. Accordingly, in order to obtain the maximum precision, accuracy, sensitivity, specificity and reproducibility of the assay, purified, well-characterized synthetic radioactive analytes are required.
Several deficiencies in radioimmunoassay methodology have been identified. First of all, it is necessary to make a physical separation of the antibody bound radiolabeled analyte from the free radiolabeled analyte. Further, the methodology is considered rather labor intensive; and the equipment required is likewise relatively expensive, is not uniformly available, and further requires the use of highly trained and skilled technicians to accurately carry out such assays. Likewise, the radioisotopically-labeled analytes are relatively unstable and expensive and pose an increasingly severe waste disposal problem owing to radiation exposure hazards associated with the commonly used radioisotopic labels. Despite these shortcomings, the use of radioimmunoassay has grown considerably.
The substantial recent growth in the use of radioimmunoassay in clinical laboratories has, however, spurred the development of variants which overcome the deficiencies of the radioimmunoassay methodology as described herein. The approaches which have been developed to overcome these deficiencies primarily involve the use of enzyme or fluorescent labels instead of radioisotopic labels, preferably coupled with conditions allowing for measuring a chemical distinction between bound and free fractions of labeled analyte which leads to the elimination of the requirement for physical separation. Immunoassays having the latter simplifying and advantageous feature are referred to as homogeneous immunoassays as opposed to heterogeneous immunoassays where physical separation is required.
Thus, homogeneous immunoassay systems have been developed which are based on the use of an enzyme-labeled analyte where the enzymatic activity of the label is decreased when complexation with the antibody occurs. Unlabeled analyte whose concentration is to be determined displaces the enzyme-labeled analyte bound to the antibody, thus causing an increase in enzymatic activity. Standard displacement or dose-response curves are constructed where increased enzymatic activity (monitored spectophotometrically using what has been termed a "substrate" which ultimately produces a unique chromophore as a consequence of enzyme action) is plotted against increased analyte concentration. These are then used for determining unknown concentrations. The following U.S. patents have been issued in the field of homogeneous enzyme immunoassay: U.S. Pat. Nos. 3,817,837; 3,852,157; 3,875,011; 3,966,556; 3,905,871; 4,065,354; 4,043,872; 4,040,907; 4,039,385; 4,046,636; 4,067,774; 4,191,613; and 4,171,244. In these patents, the label for the analyte is described as an enzyme having a molecular weight substantially greater than 5,000. Commercialization of this technology has been limited so far to applications where the analytes are relatively small in molecular size at fluid concentrations of the analyte greater than 10.sup.-10 M.
As a consequence of the limitations of the homogeneous enzyme immunoassay techniques described above, considerable effort has been devoted towards developing more sensitive homogeneous immunoassays using fluorescence. These have been primarily directed at assays for the larger sized molecules such as immunoglobulins or polypeptide hormones such as insulin. The following U.S. patents have been issued for this type of assay: U.S. Pat. Nos. 3,998,943; 3,996,345; 4,174,384; 4,161,515; 4,208,479 and 4,160,016. The label in most of these patents involves an aromatic fluorescent molecule, bound either to the analyte or to the antibody. All likewise involve various methods of quenching fluorescence through antibodies or other fluorescent quenchers so that the extent of quenching is related to the amount of analyte present in the sample.
A further type of methodology which may be described as a reactant-labeled fluorescent immunoassay involves the use of a fluorescent-labeled analyte designed so that a fluorescent product is released when it is enzymatically hydrolyzed. Antibody to the analyte portion of the molecule, however, inhibits enzymatic hydrolysis. Consequently, by the law of mass action, fluorescence is enhanced in the presence of increased analyte due to enzymatic hydrolysis of the displaced, fluorescent labeled analyte. As an example, a labeled analyte is .beta.-galactosyl-umbelliferone-sisomicin. The enzyme .beta.-galactosidase cleaves the sugar from the umbelliferone moiety which can then fluoresce. Publications which describe this methodology include: J. F. Burd, R. C. Wong, J. E. Feeney, R. J. Carrico and R. C. Boguolaski, Clin. Chem., 23, 1402 (1977); Burd, Carrico, M. C. Fetter, et al., Anal. Biochem., 77, 56 (1977) and F. Kohen, Z. Hollander and Boguolaski, Jour. of Steroid Biochem., 11, 161 (1979).
Ribonucleases are a class of widely distributed and commonly known phosphodiesterases which specifically catalyze the hydrolysis of 3'-internucleotide phosphate ester bonds of ribonucleic acids, commonly known as RNA, but not those of deoxyribonucleic acids, commonly known as DNA, or the phosphate ester bonds of simple phosphodiesters, such as, for example, bis(p-nitrophenyl) phosphate. The study of the mechanism of the hydrolysis of ribonucleic acid has been extensively recorded in the literature. See the review by F. M. Richards and H. W. Wyckoff in The Enzymes, (P. D. Boyer, Ed.), Academic Press, 3d Edition, Volume 4, pages 647-806, London and New York (1978).
Many organic compounds have been utilized heretofore for monitoring the catalytic activity of ribonuclease. Such organic compounds, or substrates, as they are commonly referred to, include ribonucleic acid itself, cyclic phosphate diesters, and monoribonucleotide compounds which exhibit the same or similar structural constraints as those expressed by the natural substrate.
Thus, for example, one method for monitoring the catalytic activity of ribonclease involves the use of a ribonucleic acid solution. That method involves monitoring a decrease in absorbance at 300 nm of a ribonucleic acid solution as a function of time, M. Kunitz, J. Biol. Chem., 164, 563 (1946). Although that method is relatively simple to conduct, it has several deficiencies; specifically, the rate of decrease of absorption is not linear, calibration of each substate solution is required, and direct monitoring of absorbance decreases at 300 nm is impractical with clinical samples.
Another method utilized for monitoring ribonuclease activity is an end-point variant of the procedure described above. In the end point variant procedure, yeast ribonucleic acid is incubated with the enzyme sample for a fixed period of time. The remaining RNA is precipitated with perchloric acid or uranyl acetate/trifluoroacetic acid, and the absorbance of the supernatant is measured after centrifugation, S. B. Anfinsen, R. R. Redfield, W. L. Choate, A. Page, and W. R. Carroll, Jour. Biol. Chem., 207, 201 (1954). However, that method is much too cumbersome for homogeneous immunoassays of the type described in the co-pending Farina et al. application primarily due to the precipitation step involved.
Yet another variation of the above procedures has been reported by R. C. Kamm, A. G. Smith, and H. Lyons, Analyt. Biochem., 37, 333 (1970). The method described therein is based on the formation of a fluorescent reaction product resulting from the reaction of the dye, ethidium bromide, with intact yeast ribonucleic acid, but not with the hydrolysis products. In that method, a fluorescent signal, which is monitored, decreases with time. However, monitoring a fluorescent signal which decreases with time is disadvantageous, as the method may result in a lack of sensitivity when only modest differences in enzyme concentration are encountered. In addition, other disadvantages are that the rate of decrease of absorption is not linear; and calibration of each substrate solution is required.
Another known substrate for monitoring ribonuclease activity is a mononucleotide substrate, cytidine 2', 3'-phosphate, E. M. Crook, A. P. Mathias, and B. R. Rabin, Biochem. J., 74, 234 (1960). In that method, an increase of absorbance at 286 nm, corresponding to the hydrolysis of the cyclic phosphate ring, is monitored over a two-hour period to measure the ribonuclease activity of the sample. This method, however, cannot be used in homogeneous immunoassay methods of the type described in the Farina et al. co-pending application because there are analyte sample interferences which occur at 286 nm. Furthermore, the distinction between the substrate and product absorbance spectra is small, with the ratio of extinction coefficients being only 1.495 at 286 nm.
Further, certain mononucleotide-3'-phosphodiesters, including, 1-naphthyl esters of 3'-uridylic, 3'-inosinic and 3'-adenylic acids have been utilized as ribonuclease substrates. These naphthyl esters have been used to differentiate substrate specificities of ribonucleases from various sources. H. Sierakowska, M. Zan-Kowalczewska, and D. Shugar, Biochem. Biophys. Res. Comm., 19, 138 (1965); M. Zan-Kowalczewska, A. Sierakowska, and D. Shugar, Acta. Biochem. Polon., 13, 237 (1966); H. Sierakowska and D. Shugar, Acta. Biochem. Polon., 18, 143 (1971); H. Sierakowska and D. Shugar, Biochem. Biophys. Res. Comm. 11, 70 (1963). As a result of ribonuclease-induced hydrolysis, the use of such substances results in the liberation of 1-naphthol which is allowed to react with a diazonium salt to form an azo compound having strong visible absorbance. This approach requires that the assay kit include a separately packaged dye forming reagent (viz.--a diazonium salt). Methods for preparing mononucleotide-3'-phosphodiesters are known. Syntheses are disclosed in R. Kole and H. Sierakowska, Acta. Biochem. Polon, 18, 187 ( 1971) and Polish Pat. No. 81969.
Still other compounds have been utilized for kinetically monitoring ribonuclease activities. Such compounds include 3'-uridylic acid phosphodiesters of 1-naphthol, 5-hydroxynaphthol, and 4-methoxyphenol, H. Rubsamen, R. Khandler, and H. Witzel, Hoppe-Seyler's Z. Physiol. Chem., 355, 687 (1974). However, the hydrolysis product is monitored directly in the ultraviolet region, at or around 280 nm, where serum interferences are expected to occur. Further, these substrates are difficult to prepare, requiring numerous steps, including lengthy chromatographic procedures.
Thus, despite the considerable number of compounds that have been developed and utilized for monitoring ribonuclease activity, there remains the need for further development which can overcome the various shortcomings of the presently known substrates.
It is, accordingly, an object of the present invention to provide novel substrates which include species that may be utilized for both direct spectrophotometric and fluorometric monitoring of catalytic activity resulting from hydrolysis of the substrate.
A further object lies in the provision of a novel substrate which is catalytically converted to product rapidly enough so that the appearance of product can be monitored kinetically over a relatively short period of time.
A still further object of this invention is to provide a novel substrate which is sensitive to ribonuclease activity even at extremely low concentrations. A related object provides a substrate capable of readily allowing detection of ribonuclease activity at low concentrations in a variety of physiological fluids such as serum, urine and the like.
Yet another object of the present invention is to provide a substrate that may be readily prepared.
A still further object provides a substrate capable of being stored in a blocked form with long term hydrolytic stability. A related object lies in providing a blocked substrate which may be readily deblocked.
A further object of the present invention is to provide a substrate which may be employed in carrying out immunoassays. A related object provides a substrate capable of use in homogeneous immunoassays.
Another object provides a substrate which may be used in carrying out homogeneous immunoassays in centrifugal fast analyzers.
These and other objects and advantages of the present invention will become apparent from the following detailed description.
While the invention is susceptible to various modifications and alternative forms, there will herein be described in detail and preferred embodiments. It is to be understood, however, that it is not intended to limit the invention to the specific forms disclosed. On the contrary, it is intended to cover all modifications and alternative forms falling within the spirit and scope of the invention as expressed in the appended claims. For example, while the use of the substrate will be principally described in connection with immunoassays, it should be appreciated that the substrate may be employed for monitoring any system having a component or components capable of hydrolyzing the substrate. Thus, the substrate may be utilized to quantitatively detect the presence of ribonuclease or peptidase. (S. Levit and M. S. Joshi, Analytical Biochemistry, Vol. 84, pp. 343-345, 1978.)